Fluorogenic and chromogenic substrate

Abstract

The present invention provides near-infrared fluorogenic and/or chromogenic formulations, methods, systems and kits. Upon contacting an azine with an oxidant and a peroxidase, the azine is converted to an azine derivative which is both visibly colored and fluorescent. This invention also provides high sensitive methods to detect biological molecules, showing better sensitivity than chemiluminescence methods. Advantageously, the formulation and reaction is free of luminol or luminol derivatives.

What is claimed is: 
     
       1. A formulation, the formulation comprising a) a first part and b) a second part:
 a) the first part comprising one or more azines; and 
 b) the second part comprising an oxidant, wherein the formulation is free of luminol or luminol derivatives and after admixing the first part and the second part in the presence of a peroxidase the formulation is both fluorogenic and chromogenic. 
 
     
     
       2. The formulation of  claim 1 , wherein the one or more azines is 10H-phenothiazine (PTA). 
     
     
       3. The formulation of  claim 1 , wherein the one or more azines is a mixture of PTA and sodium 3-(phenothiazine-10-yl)propane-1-sulfonate (PTAS). 
     
     
       4. The formulation of  claim 1 , wherein the oxidant is a member selected from the group consisting of H 2 O 2 , urea-H 2 O 2  and NaBO 3 . 
     
     
       5. The formulation of  claim 1 , wherein the first part further comprises a nucleophilic reagent. 
     
     
       6. The formulation of  claim 5 , wherein the nucleophilic reagent comprises at least one five- or six-membered ring aryl or heteroaryl, wherein the at least one five- or six-membered ring aryl or heteroaryl can be a fused aryl or heteroaryl. 
     
     
       7. The formulation of  claim 6 , wherein the nucleophilic reagent comprises an imidazoyl, a pyrazolyl, a triazolyl, a pyridyl, a phenyl, or a phenoyl moiety, which moiety is optionally fused to other rings. 
     
     
       8. The formulation of  claim 6 , wherein the nucleophilic reagent is a member selected from the group consisting of 4-(1,2,4-triazol-1-yl)phenol, 2-(1H-imidazol-2-yl)pyridine (2-IP), dimethylamino pyridine (DMAP) and 4-morpholinopyridine (MORP). 
     
     
       9. The formulation of  claim 8 , wherein the nucleophilic reagent is MORP. 
     
     
       10. The formulation of  claim 1 , wherein the formulation further comprises 2,2′-(hydrazine-1,2-diylidene)bis(3-ethyl-2,3-dihydrobenzo[d]thiazole-6-sulfonate) (ABTS) and or 3-indole acetic acid (IAA). 
     
     
       11. The formulation of  claim 1 , wherein the first part further comprises a buffer and a solubilizing agent. 
     
     
       12. The formulation of  claim 11 , wherein the buffer of the first part has a pH of about 3.5 to about 12. 
     
     
       13. The formulation of  claim 12 , wherein the buffer of the first part has a pH of about 4 to about 11. 
     
     
       14. The formulation of  claim 1 , wherein the second part further comprises a buffer. 
     
     
       15. The formulation of  claim 14 , wherein the buffer of the second part has a pH of about 3 to about 9.5. 
     
     
       16. The formulation of  claim 15 , wherein the buffer of the second part has a pH of about 4 to about 6. 
     
     
       17. The formulation of  claim 1 , wherein the first part and the second part are in separate containers. 
     
     
       18. The formulation of  claim 17 , wherein the peroxidase is a moiety having peroxidase-like activity. 
     
     
       19. A method for producing a fluorogenic and chromogenic reaction, said method comprising admixing
 a) a first part of a formulation comprising one or more azines; and 
 b) a second part of a formulation comprising an oxidant, wherein the formulation is free of luminol or luminol derivatives and after admixing the first part and the second part in the presence of a peroxidase the reaction is both fluorogenic and chromogenic. 
 
     
     
       20. A kit for preparing a fluorogenic and chromogenic reaction, the kit comprising:
 a) a first part comprising one or more azines; 
 b) the second part comprising an oxidant, wherein upon mixing the first part and the second part the admixture in the presence of a peroxidase, which is free of luminol or luminol derivatives, produces a fluorogenic and chromogenic reaction; and instructions for use.